Description of the alloreactive potential of mice rendered tolerant of H-2 antigens at birth by inoculation of semiallogeneic hematopoietic cells has been extended during the past year in two important ways. (1) Mixed lymphocyte reactivity has been assayed in adult mice, some of whom lost their tolerance spontaneously or whose tolerance had been perturbed by exposure to syngeneic, immunocompetent lymphocytes or by treatment with monocloncal anti-I-J antibodies. Animals that were initially tolerant, but whose tolerance was perturbed as a consequence of exposure to syngeneic, immunocompetent cells, developed positive MLRs; by contrast, mice whose tolerance was abolished by infusions of anti-I-J antibodies remained MLR negative, despite the fact that they rejected their test skin allografts. Thus, a significant discrepancy has emerged between the in vivo expression of alloreactivity (ability to accept/reject a skin allograft) and the in vitro measure of alloreactivity (MLR). These data strongly support the hypothesis that active suppression exists in tolerant animals, especially with respect to reactivity to class II H-2 antigens. (2) Precursor frequency assays have been developed to measure the clonal frequency of alloreactive cells responding to alloantigens in MLR and in CML. Preliminary studies of lymphoid cells from H-2-tolerant animals suggest that the frequency of MLR-responding clones is much more greatly reduced than the frequency of clones responsible for CML reactivity. We have also treated stable adult tolerant mice with monocloncal anti-I-J sera directed at host or chimeric I-J. Preliminary studies suggest that tolerance can be abolished if the anti-I-J serum is directed at the allo-I-J of the chimeric cells. This result suggests that tolerance is maintained by a process dependent upon recognition of the allo-I-J of the original donor inoculum. (LB)